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Molecular cloning and characterization of v-mos-activated transformation-associated proteins
J C Chan1, M Scanlon, H Z Zhang
1Department of Tumor Biology, University of Texas M.D. Anderson Cancer Center, Houston 77030-4009.
Abstract:
Using monoclonal antibodies we previously detected two forms of transformation-associated proteins, a 64-kDa protein and a 68-kDa protein, in temperature-sensitive 110-Moloney murine sarcoma virus-mutant-transformed rat kidney 6m2 cells. The identity and functions of the transformation-associated proteins were previously unknown. By molecular cloning techniques and immunoscreening, we have isolated two cDNA clones (34A and 79B3) that were found by Western blot analysis to code for a monoclonal anti-transformation-associated protein antibody-reactive polypeptide of approximately 58 kDa. Limited restriction enzyme mapping indicated 34A and 79B3 are two different cDNA clones. The nucleotide sequence of 34A cDNA was determined, and a search of GenBank revealed that it is identical to that of rat transin-2. The deduced amino acid sequence of 34A shares 71% sequence identity with rat transin and 41-76% identity with six human metalloproteinases. The limited restriction enzyme mapping and partial nucleotide sequencing data indicated that 79B3 may be the rat transin gene. When either 34A cDNA or 79B3 cDNA was used as a probe in Northern blot analysis, one mRNA band of approximately 1.9 kilobases was detected in 6m2 cells grown at the permissive temperature of 33 degrees C, at which the cells exhibited transformation properties, and a much lower level in 6m2 cells grown at the nonpermissive temperature of 39 degrees C, at which the cells reverted to normal phenotypes. These results suggest that at 39 degrees C, these two genes were not transcribed at the same level as at 33 degrees C. Zymogram and Western blot analysis of 6m2 cells further confirmed that the 64- and 68-kDa proteins have metalloproteinase activities and that the synthesis of metalloproteinases was also temperature-sensitive. Apparently, the two proteins we formerly designated transformation-associated proteins are members of the rat transin gene family. Therefore, within v-mos transformed 6m2 cells, the absence of transformation-associated protein (metalloproteinase) synthesis at the nonpermissive temperature was due to the absence of transcription of two rat transin genes.
Insights
Previously unidentified transformation-associated proteins in Moloney murine sarcoma virus-transformed cells were identified as metalloproteinases belonging to the rat transin gene family. Their synthesis is regulated by temperature-sensitive gene transcription.
Area of Science:
- Molecular biology
- Cancer research
- Virology
Background:
- Transformation-associated proteins (TAPs) of 64- and 68-kDa were previously detected in temperature-sensitive Moloney murine sarcoma virus-mutant-transformed rat kidney 6m2 cells.
- The identity and function of these TAPs remained unknown.
Purpose of the Study:
- To identify the molecular identity and function of the previously detected transformation-associated proteins.
- To investigate the regulation of TAP synthesis in v-mos transformed cells.
Main Methods:
- Molecular cloning and immunoscreening to isolate cDNA clones.
- Western blot, Northern blot, and zymogram analyses.
- Nucleotide sequencing and sequence homology searches (GenBank).
Main Results:
- Two cDNA clones, 34A and 79B3, were isolated, coding for a 58 kDa polypeptide reactive to anti-TAP antibody.
- Clone 34A is identical to rat transin-2; clone 79B3 is likely a rat transin gene.
- Both genes showed temperature-dependent mRNA expression, with higher levels at the permissive temperature (33°C) and lower levels at the nonpermissive temperature (39°C).
- The 64- and 68-kDa proteins possess metalloproteinase activities, and their synthesis is temperature-sensitive.
Conclusions:
- The previously identified TAPs are members of the rat transin gene family, exhibiting metalloproteinase activity.
- Temperature-sensitive synthesis of these metalloproteinases in v-mos transformed 6m2 cells is due to differential transcription of two rat transin genes.